Dataset Information

Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes

ABSTRACT: Protein ubiquitination is mediated sequentially by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2, and ubiquitin ligase E3. Uba1 was thought to be the only E1 until the recent identification of Uba6 as an alternative. To differentiate the biological functions of Uba1 and Uba6, we applied a novel orthogonal ubiquitin transfer (OUT) technology to profile their ubiquitination targets in mammalian cells. By expressing pairs of an engineered ubiquitin and engineered Uba1 or Uba6 that were generated for exclusive interactions, we identified 697 Uba6 targets and 529 Uba1 targets with 258 overlaps. Bioinformatics revealed substantial differences in pathways involving Uba1- and Uba6-specific targets. We demonstrated that polyubiquitination and proteasomal degradation of ezrin and CUGBP1 require Uba6, but not Uba1, and Uba6 is involved in the control of ezrin localization and epithelial morphogenesis. These data reveal the distinctive substrate pools for Uba1 and Uba6 and manifest non-redundant biological roles for Uba6.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Early Embryonic Cell

SUBMITTER: Duc Duong

LAB HEAD: Hiroaki Kiyokawa

PROVIDER: PXD005513 | Pride | 2017-07-31

REPOSITORIES: Pride

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Publications

Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes.

Liu Xianpeng X Zhao Bo B Sun Limin L Bhuripanyo Karan K Wang Yiyang Y Bi Yingtao Y Davuluri Ramana V RV Duong Duc M DM Nanavati Dhaval D Yin Jun J Kiyokawa Hiroaki H

Nature communications 20170130

Protein ubiquitination is mediated sequentially by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. Uba1 was thought to be the only E1 until the recent identification of Uba6. To differentiate the biological functions of Uba1 and Uba6, we applied an orthogonal ubiquitin transfer (OUT) technology to profile their ubiquitination targets in mammalian cells. By expressing pairs of an engineered ubiquitin and engineered Uba1 or Uba6 that were generated for excl ...[more]

PMID: 28134249

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Project description:Protein methylation has been implicated in many important biological contexts including signaling, metabolism, and transcriptional control. Despite the importance of this post- translational modification, the global analysis of protein methylation by mass spectrometry-based proteomics has not been extensively studied due to the lack of robust, well-characterized techniques for methyl-peptidemethyl peptide enrichment. Here, to better investigate protein methylation, we optimized and compared two methods for methyl-peptidemethyl peptide enrichment: immunoaffinity purification (IAP) and high pH strong cation exchange (SCX). Comparison of these methods revealed that they are largely orthogonal for monomethyl arginine (MMA), suggesting that the usage of both techniques is required to provide a global view of protein methylation. Using both IAP and SCX, we investigated changes in protein methylation downstream of protein arginine methyltransferase 1 (PRMT1). Together, these techniques allowed us to quantify over ~1,000 arginine methylation sites on 407 proteins. Of these methylation sites, PRMT1 knockdown resulted in significant changes to 110 arginine methylation sites on 59 proteins. In contrast, zero lysine methylation sites were significantly changed upon PRMT1 knockdown. In PRMT1 knockdown cells, 24 MMA sites exhibited both significant downregulation (ie, putative PRMT1-mediated MMA targets) and 63 significant upregulation (ie, putative PRMT1-mediated ADMA targets). PRMT1 knockdown induced significant changes in asymmetric dimethyl arginine (ADMA), consistent with being PRMT1 targets. Additionally, We also observed that PRMT1 knockdown induced significant increases in symmetric dimethyl arginine (SDMA) methylation, suggesting that loss of PRMT1 activity allows scavenging of PRMT1 substrates by Type II PRMTs. Analysis of the subcellular localization and protein function of the significantly changing methyl-proteins revealed that the majority of PRMT1 substrates are localized to the nucleus and involved in RNA and DNA binding. Taken together, our results suggest that deep protein methylation profiling by mass spectrometry requires orthogonal enrichment techniques, identify novel PRMT1 methylation targets, and highlight in order to understand the dynamic interplay between methyltransferases in mammalian cells.

Dataset Information

Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes

Publications

Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes.

Similar Datasets

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